We have developed a novel flow-through density gradient cell separation method for continuously harvesting human hematopoietic progenitor cells (1, 2), leukocytes and mouse dendritic cells. This system continuously separates a large number of cells into five fractions according to their densities. As the blood contains a huge number of red blood cells (over 1,000 times of the number of leukocytes), the pretreatment with dilution and hemolysis is usually essential for harvesting leukocytes. As the result, the cell separation time was prolonged due to the increased sample volume by the dilution which might cause cell damage and hemolysis. Therefore, we modified the present method for improving the above two points. Before the separation, Percoll density medium with the density of 1.050, which was similar to that of blood, was flowed through the sample channel instead of phosphate buffered saline before introducing the whole blood sample without diluton. And, separated cells were separated again to improve the purity. We separated basophils, which had the fewest number among leukocyte population and reported having the novel role on chronic allergic inflammation (3), under these modified conditions. A set of isosmotic Percoll media with the densities of 1.050, 1.074, 1.079, 1.090, 1.095 and 1.104 g/ml was prepared, and introduced into the channel to form a density gradient. Then the anti-coagulated whole blood was continuously fed into inlet 1, through which Percoll medium with the density of 1.050 g/ml was flowing. Harvested fractions with the density of 1.079 g/ml and 1.090 g/ml were washed, and the cell pellets were re-suspended into just 1 ml of the density medium with 1.050 g/ml. This cell suspension was fed through inlet 1 and separated under the same condition, again. Leukocyte classification of basophils in the density of 1.079 g/ml was approximately 72% and that of neutrophils in 1.090 g/ml was approximately 98% after the second run, respectively. The red blood cell counts were about 5% in each fraction. Without diluting the blood sample, the time required for cell separation was shortened and the repeating operation made possible to remove most of red blood cells. The present method might be useful for purifying some functional cells by FACS, as the preparative separation not using any antibodies and hemolytic agents. References 1. Y. Ito, K. Shinomiya, A new continuous-flow cell separation method based on cell density: principle, apparatus, and preliminary application to separation of human buffy coat, J Clin Apher. 16(2001)186-191. 2. H. Shiono, H. M. Chen, T. Okada, Y. Ito, Colony-forming assay for human hematopoietic progenitor cells harvested by a novel continuous-flow cell separation method, J. Chromatogr. A 1151(2007) 153-157. 3. H. Karasuyama, K. Obata, T. Wada, Y. Tsujimura, K. Mukai, Newly appreciated roles for basophils in allergy and protective immunity, Allergy 66 (2011) 1133-1141.